Gelatine-based materials as swabs

ABSTRACT

A swab comprising gelatine or collagen has been found to have a remarkably high recovery of microorganisms. Furthermore, the samples, such as microorganisms, spores, nucleotides and other biologically or biochemically relevant compounds can be fully recovered from the collagen-or gelatine-comprising swab. The invention thus provides a method and swab which has a high recovery of a target from a sample and furthermore a second high recovery when transferring from the swab to a medium for analysis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a U.S. national stage application ofInternational Application No. PCT/DK2003/000855, filed 11 Dec. 2003,which claims priority of Denmark Patent Application No. PA 2002 01896,filed Dec. 11, 2002. The International Application also claims thebenefit of U.S. Provisional Patent Application No. 60/432,232, filedDec. 11, 2002. All of the above applications are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

A gelatine or collagen-based material is used in the collection oftargets such as microbiological cells, mammalian cells, nucleotides andother chemical and biological molecules from an array of collectionmedia.

BACKGROUND OF THE INVENTION

Gelatine-based sponges have been used as haemostatic agents in surgicalprocedures.

Dean Jr. (U.S. Pat. Nos. 4,997,753; 4,863,856 and 4,861,714) disclosesthe use of weighted collagen microsponges for immobilising bioactivematerials.

EP 0 702 081 discloses a matrix for tissue culturing comprising twokinds of sponges.

U.S. Pat. No. 5,462, 860 discloses a culture medium for rapid growth anddetection of microbes.

Conventional methods of sampling an area for a target area comprise theuse of cotton swabs. Sample recovery from the surface is limited due tothe low recovery from the surface area to the swab and then low transferfrom the swab to a culturing medium. The level of recovery ofmicro-organisms from surfaces is critical when complying with USP/NFguidelines and EU-GMP guidelines. The present invention addresses thisproblem and provides for a dramatically improved device for sampling anarea.

SUMMARY OF THE INVENTION

A gelatine-based sponge has been found to be useful in the collection ofa variety of materials, such as microbes and mammalian cells, but alsomammalian tissue and various molecules, including nucleotides, from anarray of collection media. The sponge can be used for instance forsampling and culturing purposes. The present inventors have surprisinglyfound that the sponge has a dramatically higher recovery level of thesampled material than conventional methods. Moreover, the targetmaterial bound to the sponge can be transferred from the sponge toanother medium by an array of methods.

A first object of the invention relates to a device comprising a devicefor sampling or collecting comprising i) a swab comprising gelatine orcollagen; and ii) a support fixed to said swab.

The device or the gelatine-based sponge may be used for an array ofapplications related to the high recovery of targets from the sponge.Accordingly, a further object of the invention relates to the use of agelatine-based sponge for collection of a target from a collectionmedium comprising making contact between the gelatine-based sponge andthe medium. Furthermore, an object of the invention relates to a methodof lowering the amount of a target in a sample area comprising makingcontact between a gelatine-based sponge and at least a portion of saidsample area, such that the target adheres to the sponge. An importantand further utilisation of the surprising properties of the spongerelate to a method of qualitatively or quantitatively sampling an areafor content of a target comprising the use of a gelatine-based spongeand the steps of i) wet sampling using the gelatine-based sponge; and/orii) dry-sampling using the gelatine-based sponge. A similarly importantaspect of the invention relates to a method for culturingmicro-organisms or mammalian cells comprising adhering the cells to agelatine-based sponge and culturing the cells in a growth medium.

The invention provides a swab which has a high recovery of a target froma sample and furthermore a second high recovery when transferring fromthe swab to a medium for analysis.

DESCRIPTION OF THE INVENTION

In the present context, a target refers to any species which binds tothe gelatine-based sponge of the invention. A collection medium refersto any medium from which said targets may be collected. The term“transfer medium” is intended to mean a medium to which the collectedtarget is transferred.

In the present context, the term “recovery” is intended to mean theoverall recovery yield of a target from a collection medium to transfermedium. Thus, recovery comprises i) the collection yield of a target tothe collagen-based sponge of the present invention, as well as ii) thetransfer yield from the collagen-based sponge to a transfer medium.

A first transfer medium is, in the present context, considered to be amedium used in the collection of a target from the sponge. Suitableexamples of a first transfer medium include an enzymatic solution, or asuitable washing agent to mechanically or chemically remove the targetfrom the sponge into the medium, such as a liquid medium. A secondtransfer medium is considered to be represented by a second medium intowhich the first medium or the sample from the first medium, whichincludes the collected target, is transferred.

In the present context, a micro-organism is considered to be anyorganism selected from the group consisting of bacteria, bacterialspores, archea, yeast and fungi.

A dispersion agent is, in the present context, considered to be anyliquid agent into which targets may be dispersed following collection.

A neutral diluent is considered to be a liquid which is neutral in thecontext of the assay process, i.e. it does not interfere with thediagnostic assay being performed. In this context, therefore, a neutraldiluent is not necessarily, but can be, a diluent with neutral pH.Water, aqueous buffer solutions are suitable examples of neutraldiluents.

Ringer's solution refers to an aqueous solution comprising distilledwater and sodium chloride, potassium chloride, and calcium chloride atroughly the same concentrations as their occurrence in body fluids.

A “semi-solid surface” is a surface which is not strictly solid innature, such as mammalian tissue or any other natural and/or synthetictissue. Suitable examples of semi-solid surfaces are mammalian skin orother surfaces covered by connective tissue, such as surfaces ofmammalian organs.

In the present context, a “detergent” can be any natural or synthetic,organic or inorganic, compound or a mixture of compound used forcleaning purposes, such as for the removal of impurities or contaminantsfrom a surface.

The term “handle” in the present context is to be considered to relateto any device that can be used for gripping, and should not be construedto be limited to devices specially designed to act as a handle. By wayof an example, a stick attached to the gelatine-based sponge of theinvention can thus be used to handle the sponge, and is thereforeconsidered to represent a handle. Moreover, a tweezer or tong, whichonly temporarily connect to the sponge, is to be considered a handle.

A swab is in the present context considered to be any material used forapplying or removing material from an area or a surface. Swabbing refersto the method of applying or removing material from an area or surfaceusing said swabs.

The present invention relates to gelatine-based sponges and theirproperties with respect to binding of targets, such as micro-organismsand mammalian cells, as well as molecular species such as nucleotides,which are suitable for binding to the gelatine-based sponges. Thebinding properties of the aforementioned targets are useful for thecollection of said targets from various media. Once the target iscollected, it may be optionally be released from the sponge bymechanical, enzymatic, or chemical processes.

The target is typically selected from the group consisting of a virus, amicro-organism, a mammalian cell and an organic molecule. The organicmolecule is selected from the group consisting of a nucleotide, anucleic acid, protein or a detergent. The nucleotide is a purine- or apyrimidine-containing nucleotide, preferably ATP. The micro-organism isselected from the group consisting of bacteria, archea, bacterialspores, yeast and fungi.

The mammalian cell may be selected from the group consisting of cellsfrom blood plasma, leukocytes, erythrocytes, thrombocytes, but alsoother mammalian cells such as skin cells or any other type of mammaliancells that may be useful to collect for various diagnostic and/orcleaning purposes.

The high recovery rate, resulting from the high collection yield of atarget to the swab of the present invention, and/or the subsequent hightransfer yield from the swab to a transfer medium requires, according tothe present invention, i) a swab comprising gelatine or collagen. Theswab is typically fixed onto a handle or support

In a particularly interesting embodiment of the invention, the swab maybe selected from the group consisting of a gelatine-based sponge,collagen-based sponge, microfibrillar gelatine or microfibrillarcollagen. Preferably, the swab is a gelatine-based sponge orcollagen-based sponge, more preferably a gelatine-based sponge. Thegelatine-based sponge or collagen-based sponge material ispreferentially comprised of at least 50% gelatine or collagen,respectively, such as at least 60%, such as at least 70%, typically atleast 75%, preferably at least 80%, more preferably at least 85%, suchas at least 90%, suitably at least 95%, most preferably selected from atleast 96%, 97%, 98%, 99% gelatine or collagen, respectively, based onthe dried weight of the sponge.

In a most preferred embodiment, the swab is a gelatine sponge or acollagen sponge, more preferably a gelatine sponge. That is to say thatthe swab itself is a sponge, typically comprising at least 95%, mostpreferably selected from at least 96%, 97%, 98%, 99% gelatine orcollagen, respectively, based on the dried weight of the sponge.

The gelatine-based sponge or collagen-based sponge are characterised bytheir physical properties, which in particular may be described by thegelatine or collagen composition, pore size, reconfirmation rate, waterabsorption and digestibility of the sponge.

Without being bound to a particular theory, the pore size of the spongeis considered to influence, at least In part, the ability of the spongeis collect and transfer the targets of the invention. Moreover, the poresize influences the density of the sponge, and also has an effect on itsphysical characteristics, such as reconfirmation rates and waterabsorption.

Without being bound to a particular theory, the high recovery may be, inpart, due an appropriate degree of roughness Imparted by the surface ofthe swab, allow collection of the samples onto the surface of the swabof the invention. Furthermore, without being bound by a particulartheory, the physical properties imparted by the chemical nature of thecollagen or gelatine comprised in the swab may also, in part, by anadhesion mechanism, contribute to the high recovery rate.

Without being bound by a particular theory, it is believed thatappropriate pore size imparts, in part, an improvement to the highinitial recovery. The gelatine or collagen in the gelatine-based sponge,collagen-based sponge, microfibrillar gelatine and micorfibrallarcollagen will form or have pores with an average pore size of about 10nm to about 2 mm. Without being bound by any one theory, particularly ina non-anhydrous environment, high recovery from the collection mediummay be imparted by a form of capillary action into the swab. For thecollection or sampling of viruses, at least 10% of the pores may have apore size of less 1000 nm, such as less than 800 nm, such as less than500 nm, such as less than 400 nm. For the collection or sampling ofbacteria, at least 10% of the pores may have a pore size of less than100 μm, such as less than 80 μm, such as less than 50 μm, such as lessthan 10 μm. For the collection or sampling of fungi or red blood cells,at least 10% of the pores may have a pore size of less than 1000 μm,such as less than 800 μm, such as less than 500 μm, such as less than100 μm, such as less than 50 μm. It is within the meets and bounds ofthe skilled person to tailor the gelatine or collagen to a desired poresize and within the meets and bounds of the skilled person to select thepore size in accordance with the target.

In a typical embodiment of the invention, the gelatine or collagen ofthe gelatine-based or collagen-based sponge is of porcine origin. It isenvisaged that the invention may be adapted to include gelatine withother origins, such as gelatine of bovine, or any other mammal,including marine mammal, fish or crayfish or vegetable origin, andincluding gelatine of any other origin, such as of gelatine of organicorigin, or synthetic or semi-synthetic origin.

Reconfirmation rates represent a measure of the elasticity of thegelatine-based or collagen-based sponge. In one embodiment of theinvention, the gelatine-based sponge has a reconfirmation rate of nomore than 10 seconds, and typically no more than 5 seconds. However, itis envisaged that sponges based on gelatine from various sources willhave a wider range of reconfirmation. The reconfirmation rate istypically determined by a method based on the rate at which the spongeregains its original size and shape, as described in Example 1.

The gelatine-based sponge of the invention typically has a waterabsorption capacity which is in the range of at least 30 g/g and moretypically at least 40 g/g, as determined in Example 3. It is howeverenvisaged that the water absorption capacity of sponges based ongelatine from various sources may be in a wider range of at least 5 g/g,such as at least 10 g/g or at least 20 g/g. The determination of waterabsorption is typically performed according to USP standards.

In a further embodiment of the invention, the swab is a natural orsynthetic material, such as an absorbent material comprising gelatineparticles or collagen particles, preferably gelatine particles. Thenatural or synthetic absorbent material is essentially any materialwhich within it or upon its surface can contain loosely bound or fixed,gelatine or cartilage particles. The gelatine or collagen particles maybe entrapped by the loose or tight weave or matrix of the material or bya adhesive substance. Within this embodiment, the particles may have aparticle size in the range of about 1 μm to about 2 mm, typically fromabout 5 μm to about 1 mm, such as from about 5 μm to about 0.5 mm, moretypically about 5 μm to about 0.25 mm, preferably about 10 μm to about0.25 mm, such as about 10 μm to about 0.1 mm.

The swab has a content of gelatine particles or collagen particles,preferably gelatine particles, constituting from 1-95% wt/wt based uponthe combined dry weight of the swab and the particles, such as 2-90%,typically 5-90%. As the skilled person will appreciate, the weightcontent will depend upon the nature of the natural or syntheticmaterial.

The swab is intended for use on an array of surfaces and othercollection medium. Depending upon the use and collection medium, whetherit be industrial machinery, walls, table tops, air vents to use inequipment in conventional or micro-scale laboratories, the size of theswab will vary. Typically, the swab is of the size in the range of about1 cm×1 cm to about 15 cm×15 cm. It may be of any shape, depending on itsuse. Particular interesting is the use of collagen-based sponges orgelatine-based sponges since these are highly compressible and can beforced or squeezed into all crevices and holes.

A further aspect of the invention is directed to a kit comprising i) aswab comprising gelatine or collagen; and ii) an agent selected from thegroup consisting of a neutral diluent, an anti-microbial agent and adispersion agent. The swab may be as described above. The agent selectedfrom the group consisting of a neutral diluent, an anti-microbial agentand a dispersion agent is present in order to assist in the collectionor sampling of the target.

A further aspect of the invention relates to the use of a device or kitas desribed herein for collection of a target from a collection mediumcomprising making contact between the swab and the target.

The inventors have found that the gelatine-based sponge bindsmicro-organisms tightly but reversibly. Thus, the swab may be used forsampling for the presence of micro-organisms from a sample, such as froma surface. In such an embodiment, the micro-organism can be transferredfrom the swab by the methods of the invention. The thus collectedmicro-organisms may optionally be furthermore cultivated, which may finduses for specific purposes such as detailed characterization of saidmicro-organism. Alternatively, the swab may be used for quantitativeremoval of micro-organisms from a sample, such as a surface. In such anembodiment, anti-microbial or disinfecting agents may optionally besuitably incorporated into the swab.

It is furthermore envisaged that the swab may be adapted for use Incollecting other types of cells in addition to micro-organisms, such asmammalian cells. Such an embodiment may be realised by, e.g. collectionfrom mammalian surfaces, such as from mammalian skin or the surface ofany mammalian organ. Furthermore, the swab may be adapted for internaluse, such as during surgical operations on a mammal, and may in suchembodiments be used to collect targets from wounds or from internalorgans of a mammal, as well as surgical equipment and specialisedfurniture, walls or floors in a health clinic or a hospital, such as insurgical rooms, or in any other facility used to conduct or performsurgical procedures.

The inventors have furthermore found that the gelatine-based spongebinds certain molecules, such as purine- or pyrimidine-based nucleotidesor nucleic acids, preferentially ATP, in a reversible fashion. Thisfinding may find a useful application in that bacterial numbers havebeen estimated in foods by measuring the amount of bacterial adenosinetriphosphate (ATP). It is envisaged that the swab may more generally beadapted to collect a variety of molecular species, and thus the swab mayfind general use for collection of certain molecules.

In one embodiment, such molecules are purine- or pyrimidine-basednucleotides, such as ATP. In another embodiment, such molecules arenucleic acids. In yet another embodiment, the molecules are detergents,which may conveniently be collected from a sample, such as a surface. Inthis context, detergents can be any natural or synthetic, organic orinorganic, compound or a mixture of compound used for cleaning purposes,such as for the removal of impurities or contaminants from a surface.

Furthermore, the swab may be adapted to collect a mixture ofmicro-organisms, mammalian cells and/or molecules simultaneously from asample. In certain embodiments, it may be useful to configure thegelatine-based sponge such that one particular type of target iscollected.

The desirable property of the swab in that it is capable of adhering toa target such as micro-organisms or mammalian cells as well as molecularspecies, especially organic molecules such as nucleotides, make avariety of useful applications possible.

In a suitable embodiment of the invention, the swab is adapted so thatit comes into contact with or is attached to a support. The purpose of asupport can be that of providing a way of handling the swab withouttouching the sponge material itself, and thus avoiding contamination.This may be especially useful for embodiments in which the sponge isideally pre-sterilized, i.e. in embodiments for the collection oranalysis of micro-organisms or mammalian cells. A support may alsofacilitate the use of the swab, and allow convenient collection oftargets from various samples. A support can furthermore be invaluablefor the collection of targets from samples that may be difficult toreach or for other applications in which the swab is difficult tomanipulate without a support.

The support may be made of any suitable material for the particular usesuch as wood, natural or synthetic polymeric material, includingplastics and rubber materials, or any other organic or inorganicmaterial suitable for the particular embodiment.

The support may be of a wide variety, such as conveniently in the formof a handle. The handle may be short, such as in the range of about 1 cmto about 30 cm, such as about 3 cm to about 20 cm, preferably about 5 cmto about 15 cm. For certain applications, a handle may suitably beconsiderably larger, such as in the range of about 30 cm to severalmeters in length. The handle may be of any shape convenient for theparticular embodiment, but is typically elongated, optionally bent, withthe gelatine-based sponge attached at one end of the handle, while theother end of the handle is used for gripping and otherwise applying thegelatine-based sponge

In one suitable embodiment, in which the swab is attached to a supportin the form of a handle, the gelatine-based sponge has an oval orotherwise elongated shaped sponge positioned at the end of a stick,which serves the function of a handle. In another embodiment, the spongeis attached to a solid support, such as a circular or rectangularsupport, which in turn is positioned on the end of a handle or stick. Itshould however be appreciated that the swab may be adapted to beattached to a support in the form of a stick or handle in multipledifferent embodiments, suitably adapted for any given use of thegelatine-based sponge.

In another embodiment, the support is optionally in the form of acoating. The coating may be comprised of any suitable material, e.g.polymeric material or plastic material, or any other material suitablyused to provide a coating. In a suitable embodiment, the coating isapplied to one side of a swab of the invention which has been adapted tobe in a flat shape.

In yet another embodiment, the support is a solid material which has asuitably adapted shape, such as the shape of a disc, cube, sphere or ablock. In such embodiments, the gelatine-based sponge is preferablyattached to one side of the support, but may be attached to several orall surfaces of the support in preferred embodiments.

An embodiment in which the swab is of a cubical shape may beparticularly useful is an embodiment for collection of liquid samples orcollection from surfaces comprising a liquid coating. In suchembodiments, the swab may suitably be attached to a support, which mayoptionally be in the shape of a handle, or suitably attached to a stickor a handle.

It should be appreciated, that due to the nature of the swab material,it may be adapted to a shape for any particular use. Thus, thedimensions and shape of the embodiments wherein the invention isrealised by mounting the swab onto a support, will be adjusted asideally suited for their uses.

The swab may be attached to the support by any conventional method knownto those skilled in the art. The nature of such embodiments will dependon the particular shape of the embodiment and its intended use.

It is furthermore envisaged that the support may be in the form of aporous container, such as a crucible or otherwise suitable materialshaped such that it encloses the swab while allowing liquid and targetsto pass through the enclosing material. In such embodiments, the encasedgelatine-based sponge can be used to collect samples from a liquid. Suchcollection may suitably be performed by immersing the encased spongeinto the liquid medium, thus allowing targets in the medium to come intocontact with and bind to the gelatine-based sponge.

In embodiments of the swab in which it is adapted to collect gaseoustargets, it is preferably adapted such that the surface area and/or poresize of the sponge is maximized. Such embodiments may be realized inaccordance with any of the embodiments disclosed above, since the natureof binding of gaseous targets follows the same principles as that oftargets in a liquid medium. Gaseous targets may be any molecularspecies, which is gaseous at the temperature applied during collection,or is a liquid or a solid compound which has a vapour pressure highenough so that the target may be collected. A gaseous target is in thiscontext considered to include targets which are solid in nature,including micro-organisms and mammalian cells, but either are trapped inliquid droplets or microdroplets or form particles that may be carriedby gases, such as ambient atmosphere.

In a further aspect of the invention, gelatine powder or collagen powdermay be applied directly to a sample for collection of targets, such aswhen the sample is or found within a liquid or fluid. The gelatinepowder may in such embodiments be recovered by filtration,centrifugation or by other means known in the art.

The gelatine powder may also be enclosed by a crucible or other suitablematerial shaped such that it encloses the powder, while allowing liquidand targets to pass through the enclosing material. Such embodiments mayin particular be useful for collection from liquid media. In yet anotherembodiment, the enclosing material is permeable to gaseous targets, andthe collection of targets is realised by placing the encased powderedgelatine material in an environment containing said gaseous targets.

Methods used for collecting targets from a surface using the swab of thepresent invention include techniques such as swab techniques andcount-tact techniques. Swab techniques involve in principle a mechanicalswiping or swabbing of a target, such as a surface, and are well knownto those skilled in the art. Count-tact techniques involve the use of aspecially designed instrument, as disclosed in Example 6.

In one embodiment of the invention, the gelatine-based sponge,optionally attached to a support, is used to collect targets from asample. The target may be selected from the group comprising amicro-organism, mammalian cell, but may also be mammalian tissue, oralternatively a molecular species, such as a nucleic acid or a purine-or pyrimidine-based nucleotide, preferentially ATP. The micro-organismmay be selected from the group consisting of bacteria, archea, bacterialspores, yeast, or fungus. The mammalian cell can be of any mammaliancell type, but may in particular be selected from the group consistingof cells from blood plasma, leukocytes, erythrocytes, thrombocytes,epithelial cells, skin cells or any other mammalian cell type that maybe useful to collect for various diagnostic and/or cleaning purposes.

In a preferred embodiment, targets are collected from a surface using acombination of wet-sampling and dry-sampling. In this context,wet-sampling is considered to comprise the use of a gelatine-basedsponge of the invention, which optionally has been attached to a supportand has been pre-wetted with a suitable neutral diluent or a dispersingagent. Such a diluent or dispersion agent serves the purpose offacilitating the recovery of the targets from the surface, while notinterfering with the assay. The diluent and dispersion agents may be anysuitable aqueous solution, optionally including salts or other agentsnot toxic or otherwise chemically or biologically harmful for thetargets to be collected, such as saline, saline peptone, buffered salinepeptone, Ringer solution and an organic or inorganic buffer, optionallycontaining inorganic salts. The diluent or dispersion agent may alsooptionally contain growth media suitable for the micro-organism ormammalian cell type being collected, for assays directed towards suchtargets. The sponge may also optionally be presterilized. Collection oftargets is realized by swiping the surface with at least one suchpre-wetted sponge, followed by swiping of said surface with at least onedry gelatine-based sponge. The purpose of the dry swiping is to recoveras much as possible of remaining liquid and target from the surface.

Collection of targets may, in other embodiments, be realised bywet-sampling or dry-sampling alone. The choice of method to use willvary depending on the sample type to be assayed and the target type tobe collected.

Targets, including micro-organisms, mammalian cells and/or moleculescollected in the swab are typically transferred from the swab. Transferof such collected targets comprises removing or unbinding such targetsfrom the sponge into a suitable medium. In a preferred embodiment, thisis accomplished by placing the gelatine-based sponge in a mediumcomprising a solution capable of digesting the gelatine-based sponge.Digestion of the sponge may be realized by chemical and/or enzymaticmethods, preferably using enzymes such as proteases, more preferablyusing proteases such as alcalase or pepsin. Digestion by chemical meansmay comprise using mineral or carboxylic acids, or bases, in appropriateconcentration not to denature the target. In one embodiment, digestioncomprises using a mixture of at least one enzyme, and may optionallyinclude a mineral acid or a base, and optionally inorganic salts as wellas organic or inorganic buffering agents. The temperature suitable forrecovery of targets from the sponge will be highly dependent on themethod used. In embodiments wherein transfer is realised using enzymes,the experimental temperature will be adjusted so as to maximise theefficiency of digestion for the particular enzyme in the context of thecomposition of the digestion medium employed.

In general, transfer of targets from the gelatine-based sponge of theinvention may be realised by any technique known in the art whichreleases said target from the sponge. Thus, this may occur by changes inconditions such as pH or temperature, or by adding salts, chaotrophicagents or organic solvents. Transfer from the sponge may also optionallyinclude mechanical action, such as that generated by rubbing or shakingthe sponge, or by other mechanical means facilitating the unbinding oftargets from the sponge. Transfer from the sponge may furthermore berealized by washing of a target from the gelatine-based sponge.

It is envisaged that preferred embodiments of the invention will includedigestion methods for releasing micro-organisms and/or mammalian cellsfrom the sponge, since micro-organisms and mammalian cells are ingeneral sensitive to changes in conditions. However, there may beexceptions to this, and in particular it is envisaged that for certainapplications it will be useful to use changes in experimental conditionsfor recovery of certain microorgansisms or mammalian cells, inparticular extremophiles, which are organisms adapted to withstand harshconditions of pH, salt, temperature and/or organic solvents. Recovery ofbound molecules to the sponge may be accomplished by any of theaforementioned techniques, depending on any given embodiment of theinvention and the type of molecule bound.

Micro-organisms or mammalian cells recovered from the gelatine-basedsponge may optionally be further isolated using membrane filtration,wherein the membrane filter has properties such that it allows solventand small molecules to pass through the filter, while whole cells andmicro-organisms do not. In a typical embodiment, such a filter has apore size of less than 1 μm, such as less than 0.8 μm, such as less than0.6 μm, more typically less than 0.45 μm, such as less than 0.2 μm.

In another embodiment of the invention, the gelatine-based sponge isused to disinfect a sample, such as a surface. In such an embodiment,the combined action of the sponge, which acts to remove targets, in thiscase micro-organisms and/or mammalian cells, from a sample, andoptionally an anti-microbial or a disinfecting agent, facilitates theeffective removal of said micro-organisms or mammalian cells from thesample, thus rendering it sterile. The sponge is in such embodimentspreferably sterilised by methods known in the art, such as by heatand/or radiation, and optionally pre-treated with an anti-microbial ordisinfecting agent. Such an agent is preferably a liquid, such as analcohol, an aqueous solution comprising an alcohol or other liquid agentwhich kills micro-organisms or mammalian cells, but may be any compoundwhich facilitates the sterilization procedure. Embodiments forsterilization of samples may be realized by packaging individualgelatine-based sponges, optionally attached to a support and optionallypretreated with a sterilizing agent, individually into sealed packages,which are ideally intended for single use.

In another embodiment of the invention, viable micro-organisms ormammalian cells which have been trapped on the gelatine-based sponge canbe cultured. Culturing in general requires contacting a micro-organismor mammalian cell and a suitable growth medium. The growth medium may inthe form of a liquid; alternatively, it is in the form of solid agar.The growth medium is comprised of components well known to those skilledin the art. Realisation of said culturing can be accomplished byconventional techniques, including:

-   1. Contacting the gelatine-based sponge, optionally attached to a    support, and a liquid or a solid growth medium. In one such    embodiment, a suitably shaped gelatine-based sponge positioned at    the end of a stick is used to collect targets from a surface, and    subsequently allowed to come into contact with a growth medium, such    that micro-organisms or mammalian cells collected by the sponge are    transferred to the growth medium.-   2. Injecting a liquid growth medium, optionally agar-containing,    into the gelatine sponge, thus providing conditions for in situ    growth of the bound micro-organisms or mammalian cells in the    gelatine-based sponge.-   3. Transferring the gelatine-based sponge to a container with liquid    growth medium, thus allowing for culturing of the entire population    of bound micro-organisms or mammalian cells in said growth medium.

It should be appreciated that the culturing of collected micro-organismsor mammalian cells may be preceded by a step in which bound cells havebeen unbound or by other means released into a medium. In suchembodiments, said medium is considered to be a first transfer medium.The medium can be any liquid suitable for the application, such as aneutral diluent, a dispersion agent, or a growth medium. Unbinding ofbound micro-organisms or mammalian cells may be realized by any of themethods described herein, including enzymatic and/or chemical digestionof the gelatine-based sponge, and may include mechanical transfer intosaid first transfer medium. The thus unbound micro-organisms ormammalian cells are subsequently transferred to a second transfermedium, which is ideally comprised of a liquid or solid growth medium.The transfer to a second transfer medium may be partial, i.e. a samplefrom said first growth medium is transferred to said second transfermedium. Alternatively, the transfer is complete, i.e. the entire volumeof the first transfer medium is transferred Into the second tranfermedium.

Culturing of micro-organisms or mammalian cells collected by thegelatine-based sponge of the invention may in particular be useful forfurther characterization or production of the collected micro-organismsor mammalian cells. Preferred embodiments of the gelatine-based spongemay, for example, be used for qualitative determination of themicrobiological and/or mammalian cell composition of a target populationcollected from a sample. In such embodiments, the sample may, forexample, be a surface in a food production line, such as meat or fishprocessing lines, or from other surfaces such as floors, walls, orequipment used in such processing lines. Alternatively, the sample maybe a surface from equipment, specialized furniture or walls or floorsfrom a health clinic or hospitals, such as surgical rooms. The samplemay also be collected from an open wound, from the surface of aninternal organ or it may be comprised of any mammalian tissue. Inprinciple, however, the gelatine-based sponge may be adapted forcollecting and culturing targets from any sample, from which it isuseful to determine the microbiological and/or mammalian cell content.

In one embodiment, the collection medium is a solid surface from whichtargets may be collected. In a second embodiment, the medium is aliquid, from which targets may also be collected. In such an embodiment,the high water absorption capacity of the gelatine-based sponge is auseful characteristic, as it allows collection of large volumes ofwater. The liquid medium may be located on a surface, for example incavities on the surface. The sponge therefore can be adapted to beuseful for the collection of targets from a wide variety of sources,such as manufacturing devices in food manufacturing, processing plantsfor meat and/or fish products, medical devices, as well as themanagement and cleaning of wounds, such as surgical wounds.

It is furthermore envisaged that the gelatine-based sponge may beadapted for use in collecting targets from other types of samples ofliquid and/or gaseous nature.

In principle, collection may be from a solid surface irrespective of thematerial from which the surface is comprised, such as natural orsynthetic surfaces, of organic or inorganic material. Furthermore,targets may be collected from semi-solid surfaces, such as mammaliansurfaces and mammalian tissue, including mammalian skin and the surfaceof mammalian organs.

EXAMPLES

The following methods and examples illustrate how the gelatine-basedsponge of the present invention may for specific uses be adapted for thecollection and recovery of bacterial spores from a stainless steelsurface. Recovery yields, i.e. the overall yields for the transfer fromthe surface to the sponge and the subsequent transfer from the sponge toa medium, are very high which is an illustration of the usefulness ofthe gelatine-based sponge for the collection and transfer of targetsfrom a sample, such as a stainless steel surface.

These methods and examples should however only be understood as examplesof useful embodiments of the present invention, and in no way limitingfor its adaptation for other use.

Example 1 Determination of Reconfirmation time of Gelatine-Based Sponges

The purpose of this method is to determine the reconfirmation rate of agelatine-based sponge. The method comprises soaking the sponge, andsubsequently squeezing it. The appearance of the native shape of thesponge is monitored as a function of time, and the time that lapsesuntil the sponge has reached its native shape is termed thereconfirmation time.

The method comprises the following steps:

-   1. Cut a suitable piece of absorbable gelatine-based sponge,    approximately 1×1 cm, and thoroughly soak it in water at room    temperature.-   2. Remove the sample from the water, and squeeze it until it is flat    and no more air bubbles or drops of water can be pressed out.-   3. Place the sample in a beaker filled with water at room    temperature and measure the time (in seconds) until the sample has    gained its former size and shape.-   4. Repeat the test twice and report the result as the average of    three determinations.

Example 2 Determination of Digestability of Gelatine-Based Sponges usingPepsin

Purpose: To Determine the Digestion Time of a Gelatine-Based Sponge byEnzymatic Means using Pepsin.

Reagents used in the Method:

-   Milli-Q-water-   Pepsin(1 :3000)-   Hydrochloric acid dilute, Ph. Eur.-   Pepsin solution 1%:    -   Accurately weigh 20.0 g of pepsin and dissolve in 100 ml        hydrochloric acid dilute, using a 2000 ml volumetric flask. Add        water to volume and mix.        Apparatus:-   Metallic filament basket, 6 cm ø, 1 mm openings.-   Thermostated water bath    Method:-   1. Transfer 100 ml of the pepsin solution (1%) to a 250 ml beaker.    Add a magnet and place the beaker in a water bath, previously warmed    to 37° C. on a magnetic stirrer.-   2. Cut off a piece of absorbable gelatine sponge weighing 50±5 mg,    and place it in a beaker of water. Knead gently between the fingers    until the gelatine sponge is thoroughly wet, and until all the air    has been removed, taking care not to break the tissue.-   3. Lift from water and squeeze gently to remove any excess water.-   4. Place the wetted sample in a metallic filament basket in the    beaker, and start the timer.-   5. Watch until the piece of absorbable gelatine sponge is entirely    dissolved, stop the timer, and note the consumption of time.-   6. Repeat the test twice—a total of three times and calculate    average.    Alternative Method for a Gelatine Powder-   1. Transfer 100 ml of the pepsin solution (1%) to a 250 ml beaker.    Add a magnet and place the beaker in a water bath, previously warmed    to 37° C. on a magnetic stirrer.-   2. Prepare a sample weighing out approximately 50 mg±5 mg.-   3. Place the sample directly in the beaker.-   4. Watch until the absorbable gelatine powder is entirely dissolved,    stop the timer, and note the consumption of time.-   5. Repeat the test twice—a total of three times and calculate    average of three determinations.

Example 3 Determination of Water Absorption of Gelatine-Based Sponges

Purpose: To determine the amount of water that a gelatine-based spongecan absorb. The sponge is expected to absorb several times its ownweight of water on a weight to weight basis.

Method: According to USP Method “Absorable Gelatine Sponge: Waterabsorption”. A total of 6 determinations on 6 different pieces ofgelatine-based sponge are performed.

Example 4 Size Measurements of a Celatine-Based Sponge

Purpose: The dimensions weight, height, length, width, centre hole anddiameter of the gelatine sponge are measured on 6 samples; one series ofmeasurements Is performed for each of the 6 samples. The average of the6 measurements is reported. The density is calculated.

Apparatus: A caliper, Mitutoyo 500-Series or similar.

-   -   A ruler specifically made for the determination of length and        width of absorbable gelatine sponge film.    -   Balance, Mettler AK 160 or a balance with similar accuracy.

Method and Calculations: The wanted measurements are made with a caliperor a ruler. The average of 6 measurements performed on 6 samples isreported (in mm).

The weight of the sponge is measured. The average of 6 measurementsperformed on 6 samples is reported (in 0.001 g). The density of theabsorbable gelatine sponge, anal is calculated in the following way:

${\frac{4*{weight}}{\pi*{length}*\left( {D^{2} - d^{2}} \right)} = {density}},{{mg}\text{/}{mm}^{3}}$where

-   D=diameter of the sponge.-   d=diameter of the pore in the sponge.

Example 5 Sampling Protocol from a Stainless Steel Surface using WetSampling and Dry Sampling

Purpose: To sample a stainless steel surface using a combination of wetsampling and dry sampling.

Materials:

-   A gelatine-based sponge.-   Saltwater-peptone solution-   Alcalase solution-   A stainless steel surface with a 24 cm² contact area.-   Stomacher bags    Method:-   1. Swab the stainless steel sheets horizontally from left to right    covering the 24 cm² contact area using a gelatine sponge swab    moistened with saltwater-peptone solution.-   2. Swap the stainless steel sheets vertically from top to bottom    covering the 24 cm² contact area using a gelatine sponge swab    moistened with saltwater-peptone solution.-   3. Swab the stainless steel sheets horizontally from left to right    covering the 24 cm² contact area using a dry gelatine sponge swab.-   4. Swap the stainless steel sheets vertically from top to bottom    covering the 24 cm² contact area using a dry gelatine sponge swab.-   5. Place the swabs in stomacher bags.-   6. Add the digesting solution to each stomacher bag.-   7. Homogenize the stomacher bags using a stomacher.-   8. Incubate the stomacher bags at 36° C. until the swabs have been    dissolved.-   9. Extraction of the spores is carried out using membrane    filtration.

Example 6 Swabbing by Count-Tact Applicator

Introduction. The Count-Tact applicator standardizes surface testing, byapplying a uniform pressure of 500±50 g for 10±1 seconds (draft EuropeanStandard: CEN/TC 243).

Description. The applicator is composed of two plastic elements:

-   -   the base, which holds the Count-Tact plate in position,        consisting of a push-button device mounted on a calibrated        spring;    -   a unit clipped onto the base, containing the electronic timer,        the audible beep mechanism and batteries.

The method involves the following steps:

-   1. Slide the Count-Tact plate into position (lid facing outwards)    under the two transparent clips fixed on the bottom of the base.-   2. Remove the lid from the Count-Tact plate.-   3. Hold the applicator in position against the surface where the    sample is to be taken, without moving it (check that the surface is    not damp).-   4. After 10 seconds of contact with the surface, the audible beep    will sound. Remove the applicator from the surface. Place the lid    back on the plate, and remove the plate from the applicator (do not    forget to clean the surface where the sample was taken in order to    remove any possible traces of agar).-   5. For incubation of the plates, refer to the Count-Tact technical    sheet and the Count-Tact irradiated sheet.

Example 7 Validation Protocol from Polished Stainless Steel

Introduction. This protocol describes the validation of a method that isused for the microbiological sampling from polished stainless steel,which has been cleaned with isopropanol 70%.

The validation protocol is carried out in order to define the accuracyand precision of the method, and the recovery yield of micro-organismsfrom the type of material used in this test, using the described methodof sampling.

This current test does apply whenever microbiological sampling frompolished stainless steel cleaned with isopropanol is carried out inaccordance with the current described sampling procedures.

Validation Parameters.

Selectivity

Bacillus spores are used as markers of microbiological activity on thestainless steel. Because the spores are incubated at 55° C., it isconsidered unlikely that any microbiological contamination willinfluence the results during the validation test. Thus, selectivity isnot relevant for this test.

Precision (Repeatability) and Accuracy (Recovery)

Assessed by replicate extraction and analysis of swab samples using thesame analyst, same equipment and same reagents within the same day,using

-   -   2 concentration levels (5 and 25 spores); 1 surface material; 2        wipings of each surface material (pooled to one sample for        analysis); Total of six replicates (n=6); Therefore, a total of        12 samples are analyzed.        Linearity

Established using a three-point calibration curve, covering ranges from5 to 50 spores. Test is carried out in six replicates, using threeconcentration levels (5, 25 and 50 spores), and 2 wipings of eachsurface material, pooled for analysis.

Intermediate Precision

Established by replicate analysis on different days, using same reagentbatches, same equipment, different analysts. Demonstrated using 2concentration levels, 5 and 25 spores, 1 surface material and twowipings (pooled for analysis), and six replicates by three analysts onthree days. Therefore, a total of 108 samples.

Equipment.

Incubators, autoclave, sterile Drigalski spates, sterile gloves,gelatine swabs, mixer, membrane filtration equipment, colony counter,sterile stomacher bags.

Materials.

Spore suspension (1*10⁶ spores/0.1 mL ethanol) of Bacillusstearothermophilus spores in 40% ethanol. Test samples of surfacematerial (polished stainless steel). Template for swabbing area (6×4cm=24 cm²)

-   Tryptic Soy Agar-   Sterile Filter-   Sterile 50 mL syringe-   Disposable sterile pipette tips-   Count-Tact applicator with contact plates containing TSA, Tween and    Lecithin.    Chemicals-   Saltwater-peptone solution-   Ethanol 96%-   Elga water-   Alcalase solution    Preparation of Test Validation Solutions-   Positive control

Class A: Spore suspensions containing 5 spores/313 μL is used aspositive control. 0,100 ml spore suspension (1,6*10⁶ spores/0,1 ml) isdiluted in 9,90 ml 40% ethanol 3 times to give 1,6 spores/0,1ml in thefinal suspension, which corresponds to 5 spores/1,6spores=3,125×0,1ml=0,3125 ml spore suspension to each contact sheet (24cm²).

Class B: Spore suspensions containing 25 spores/63μL is used as positivecontrol. 0,100 ml spore suspension (1,6*10⁶ spores/0,1 ml) is diluted in19,90 ml 40% ethanol 2 times to give 40 spores/0,1 ml in the finalsuspension, which corresponds to 25 spores/40 spores=0,625×0,1 ml=0,0625ml spore suspension to each contact sheet (24 cm²).

Negative control

-   Sterile ethanol is used as negative control    Linearity

Three spore suspensions a) containing 5 spores, b) containing 25 sporesand c) containing 50 spores will be used for the linearity study.

-   a) 0,100ml spore suspension (1,6*10⁶ spores/0,1 ml) is diluted in    9,90 ml 40% ethanol 3 times to give 1,6 spores/0,1 ml in the final    suspension, which corresponds to 5 spores/1,6 spores=3,125×0,1    ml=0,3125 ml spore suspension to each contact sheet (24 cm²).-   b) 0,100 ml spore suspension (1,6*10⁶ spores/0,1 ml) Is diluted in    19,90 ml 40% ethanol 2 times to give 40 spores/0,1 ml in the final    suspension, which corresponds to 25 spores/40 spores=0,625×0,1    ml=0,0625 ml spore suspension to each contact sheet (24 cm²).-   c) 0,100 ml spore suspension (1,6*10⁶ spores/0,1 ml) is diluted in    19,90 ml 40% ethanol 2 times to give 40 spores/0,1 ml in the final    suspension, which corresponds to 50 spores/40 spores=1,25×0,1    ml=0,125 ml spore suspension to each contact sheet (24 cm²).    Sample Preparation.    Sampling Using Swab Technique.    -   Spore suspension is applied to each stainless steel sheet    -   Ethanol from the spore suspension on the stainless steel sheets        is allowed to evaporate    -   1,0 ml alcalase solution is diluted in 90 ml saltwater peptone        solution.    -   put stomacher bag in matching stomacher bag clip    -   swab the stainless steel sheets horizontally from left to right        covering the 24 cm² contact area using a swab moistened with        slatwater-perpone solution.    -   swab the stainless steel sheets horizontally from top to bottom        covering the 24 cm² contact area using a swab moistened with        slatwater-perpone solution.    -   swab the stainless steel sheets horizontally from left to right        covering the 24 cm² contact area using a dry swab.    -   swab the stainless steel sheets horizontally from top to bottom        covering the 24 cm² contact area using a dry swab.    -   place the swabs in stomacher bags.    -   add the diluted alcalase solution to each stomacher bag.    -   homogenize the stomacher bags using a stomacher.    -   incubate the stomacher bags at 36C. until the swabs have been        dissolved (for at least 1 hour and maximum 2,5 hours).    -   extraction of the spores is carried out using membrane        filtration        Sampling Using Count-Tact Applicator    -   use gloves    -   spore suspension is applied to each stainless steel sheet    -   ethanol from the spore suspension on the stainless steel sheets        is allowed to evaporate    -   Count-Tact applicator is used on the stainless steel plates

Negative controls are also added to the stainless steel sheets. Positivecontrol samples are not added to stainless steel sheets, but are addeddirectly to the Count-Tact application plates.

Incubation

All samples are incubated at 55C.±2C. for at least 1 day to a maximum of7 days.

Cleaning of Surface Material and Templates.

Surface material and templates should be cleaned after each use andre-use:

-   -   rinse the surface by wiping with laboratory paper that has been        soaked in ethanol    -   sterilization by autoclave at 121C. for not less than 20 minutes        Calculations

-   Recovery %=sample (CFU)*100/added spores, where

-   sample=number of spores from Accuracy Test Solutions and

-   Added=number of spores in Accuracy Control Samples    Validation Procedures    Recovery/Precision/Accuracy

Recovery/Precision and Accuracy is investigated using stainless steeland performed in accordance with the methods outlined above. This testwill be used for the calculation of the microbiological recovery fromthe test samples. Batches of reagents and equipment will not be alteredsince the change in these parameters is estimated to have little or noinfluence on the final test results.

Procedure

One analyst will contaminate six stainless steel plates with approx. 5and 25 bacillus spores each.

Each stainless steel sheet is swabbed quadruplicate or sampled using theCount-Tact applicator

The samples are incubated at 55±2C. for at least 1 day to a maximum of 7days and counted.

The tests will be carried out in accordance with the method describedabove.

Recovery/Accuracy

Recovery should be higher than 20% compared to positive controls.

The lowest recovery sets the limit (the lowest averag recovery at anygiven spore concentration −5 to 25 spores/24 cm²).

Precision

-   -   calculate mean and % RSD on replicate analysis    -   % RSD should be less than 10%        Linearity        Procedure

Prepare six identical samples using the spore suspensions a), b) and c)defined above. The samples will be used to establish recoveryefficiencies for these levels of spores and used for the subsequentlinear regression.

Evaluation

-   -   recovery will be calculated for each level of spores and        regression will be made from 5 to 50 spores.    -   calculate the regression parameters of the standard curves and        report slope, intercept and correlation coefficient of each        curve.    -   the correlation coefficient should meet the criterial of        r²>0,940.        Intermediate precision        Procedure

Same as for precision described above on three following days, usingthree different analysts.

Evaluation

-   -   calculate recovery of spores    -   calculate mean and % RSD on replicate analysis for each day    -   calculate mean and % RSD using all days (intermediate precision        day 1, day 2 and day 3)    -   % RSD should be less than 15%

Example 8 Validation of Sampling from Polished Stainless Steel

Purpose

To validate the methods used for microbiological sampling from polishedstainless steel that has been cleaned with 70% isopropanol, referring tothe validation protocol of Example 7.

Introduction.

The validation study is carried out in order to define the accuracy,precision and linearity of methods used for microbiological samplingfrom surfaces and to estimate the recovery efficienty of micro-organismsfrom the type of material used in the test, using the described methodsof sampling. In the present study the sampling was performed frompolished stainless steel cleaned with 70% isopropanol. The rationale forsampling is that this is a common material used for equiment production.The method tested were sampling by swab technique and sampling bycount-tact technique. The sampling was carried out on surfaces with anapplied number of bacterial spores equal to the USP, NF guideline ofclass 10,000 production equipment and the EU-GMP guideline formicrobiological purity of class 10,000 production equipment.

Results

Recovery

Calculation of the recovery using the swab technique (Table 1) showedvariations between analysts when applying 5 spores (40% to 175%) andvariations when applying 25 spores (73% to 105%). The average recoveryfor all analysts using the swab technique were calculated to 80% whenapplying 5 spores and 91% when applying 25 spores.

Calculation of recovery using the count-tact technique (Table 2) showedvariations between analysts when applying 5 spores (54% to 88%) andvariations when applying 25 spores (36% to 57%). The average recoveryfor all analysts using the count-tact technique were calculated to 74%when applying 5 spores and 45% when applying 25 spores.

Precision

Calculations of relative standard deviation in percent (RSD %), whenusing the swab technique (Table 1) showed variations between analysts(34% to 70%) when applying 5 spores and variation (30% to 57%) whenapplying 25 spores. Using the count-tact technique (Table 2) RSD% wasbetween 41% and 90% when applying 5 spores and between 20% and 65% whenapplying 25 spores.

Intermediate Precision

Intermediate precision using the swab technique (Table 1) was 64% whenapplying 5 spores and 43% when applying 25 spores. Intermediateprecision using the coun-tact technique (Table 2) was 77% when applying5 spores and 54% when applying 25 spores.

TABLE 1 Sampling results using the swab technique Spores applied(calculated, not Assay (spores/24 cm²) actual)¹⁾ 5 25 50 Mean (cfu/24cm²) Analyst 1³⁾ 5 21 ²⁾ Precision (RSD %) 42 57 Recovery (%) 175 105Mean (cfu/24 cm²) Analyst 2³⁾ 3 15 ²⁾ Precision (RSD %) 70 30 Recovery(%) 59 73 Mean (cfu/24 cm²) Analyst 3³⁾ 2 17 29 Precision (RSD %) 34 3239 Recovery (%) 40 94 95 Mean (cfu/24 cm²) Analyst 1, 2, 3 3 18 ²⁾Precision (RSD %) 64 43 Recovery (%) 80 91 ¹⁾The number of sporesapplied was calculated to be 5, 25 or 50. The actual amound of sporesapplied was used for calculating recoveries. ²⁾One analyst only carriedout sampling from surfaces with 50 spores applied by the sole purpose ofinvestigating linearity ³⁾Each analyst completed the sampling andanalysis in six replicates.

Linearity from 5 to 50 spores:

-   Correlation coefficient (R)=0,9995; R²=0,9990; Intercept=−2,3;    Slope=1,0    Linearity

Linearity was calculated by regression between applied sporeconcentrations of 5, 25 and 50 spores by one analyst. The defined levelof acceptance was a R²>0,9400. The calculated R² was 0,9990 when usingthe swab technique and 0,9989 when using the count-tact technique.

TABLE 2 Sampling results using the count-tact technique Spores applied(calculated, not Assay (spores/24 cm²) actual)¹⁾ 5 25 50 Mean (cfu/24cm²) Analyst 1³⁾ 4 14 20 Precision (RSD %) 70 20 24 Recovery (%) 88 4540 Mean (cfu/24 cm²) Analyst 2³⁾ 2 7 ²⁾ Precision (RSD %) 41 44 Recovery(%) 79 36 Mean (cfu/24 cm²) Analyst 3³⁾ 2 13 ²⁾ Precision (RSD %) 90 65Recovery (%) 54 57 Mean (cfu/24 cm²) Analyst 1, 2, 3 3 11 ²⁾ Precision(RSD %) 77 54 Recovery (%) 74 45 ¹⁾The number of spores applied wascalculated to be 5, 25 or 50. The actual amound of spores applied wasused for calculating recoveries. ²⁾One analyst only carried out samplingfrom surfaces with 50 spores applied by the sole purpose ofinvestigating linearity ³⁾Each analyst completed the sampling andanalysis in six replicates.

Linearity from 5 to 50 spores:

-   Correlation coefficient (R)=0,9989; R²=0,9979; Intercept=−2,4;    Slope=0,4    Discussion    Recovery

The level of recovery of microorganisms from surfaces is critical whencomplying with USP/NF guidelines and EU-GMP guidelines. The bacterialrecovery using both the swab technique and the count-tact technique asdescribed in the protocol was better than anticipated when recoveringmicroorganisms from the samples with a known microbiologicalcontamination rqual to the USP/NF guideline and the EU-GMP guideline.The average (for all analysts) lowest recovery was 80% for the swabtechnique and 45% for the count-tact technique. For the chosen samplingmethod used on stainless steel this recovery should be used to estimatethe actual amount of microorganism on the surface.

Precision

The relative standard deviation (% RSD) on replicate analysis (for asingle analyst) was larger than first anticipated. The comparison of the% RSD for the positive controls and the assay however showed that high %RSD is mainly caused by the analysis of the samples and not by the useof count-tact or swab technique. It should have been expected not toachieve a % RSD corresponding to chemical analysis, since it isgenerally known that % RSD is much larger for microbiological analysisthan chemical analysis.

Intermediate Precision

The relative standard deviation (% RSD) on replicate analysis (betweenanalysts) was larger than first anticipated. The comparison of the % RSDfor the positive controls and the assay however showed that the high %RSD is mainly caused by the analysis of the samples and not by the swabor count-tact techniques used. It should have been expected not toachieve a % RSD corresponding to chemical analysis, since it isgenerally known that % RSD Is much larger for microbiological analysisthan chemical analysis.

The average % RSD calculated for the lowest recoveries given above was64% for the swab technique and 54% for the count-tact method. To correctfor the large variations on the analysis the % RSD should be taken intoaccount when estimating the actual number of microorganisms on asurface.

Conclusions

The results show that the methods described above can be used forsampling of microorganisms from polished stainless steel in an overallsatisfactory way and show no reason not to expect the method to beamenable to all surfaces. The level of recovery of micro-organisms fromsurfaces is critical when complying with USP/NF guidelines and EU-GMPguidelines. The bacterial recovery using both the swab technique and hecount-tact applicator as described in the protocol was better thananticipated when recovering micro-organisms from samples with a knownmicrobiological contamination equal to the USP/NF guideline and theEU-GMP guideline.

1. A single-use device for sampling and recovering a target, said devicecomprising a sterile swab and a handle attached to said swab, whereinsaid swab is a gelatine-based sponge, and wherein said device iscontained in a sealed package.
 2. The device according to claim 1,wherein the gelatine-based sponge has a water absorption capacity of atleast 30 g/g as determined by USP method “Absorbable Gelatine Sponge:Water Absorption”.
 3. A kit comprising: i) a swab and a handle attachedto said swab, wherein said swab is a gelatine-based sponge; and ii) aneutral diluent selected from the group consisting of saline, salinepeptone, buffered saline peptone, Ringer solution and an organic andinorganic buffer.
 4. A method for collecting a target from a collectionmedium comprising: i) providing a device according to claim 1; ii)making contact between the swab of the device according to claim 1 andthe target to transfer said target from the collection medium to theswab of the device; and iii) transferring said target from the swab ofsaid device to a transfer medium to thereby recover said target fromsaid collection medium.
 5. A method for sampling an area for a targetcomprising: i) swiping the surface of the area with a firstgelatine-based sponge pre-wetted with a neutral diluent or dispersingagent; followed by ii) swiping said surface with a dry secondgelatine-based sponge; and iii) transferring said target from said firstand second sponges to a transfer medium. thereby recovering the targetfrom said surface.
 6. A method of lowering the amount of a target in asample area comprising: (i) providing a device according to claim 1,(ii) making contact between the swab of the device according to claim 1and at least a portion of said sample area, so that an amount of thetarget adheres to the swab of the device, and (iii) transferring theadhered target from the swab of said device to a transfer medium.
 7. Themethod according to any one of claims 4, 5 and 6, wherein the target isselected from the group consisting of a virus, a microorganism, amammalian cell and an organic molecule.
 8. The method according to claim7, wherein the organic molecule is selected from the group consisting ofa nucleotide, a nucleic acid, a protein and a detergent.
 9. The methodaccording to any one of claims 4, 5 and 6 wherein said transferring stepincludes digestion of said gelatine swab.
 10. The method according toany one of claims 4, 5 and 6 wherein said transferring step includeswashing said target from the gelatine swab.
 11. The method according toclaim 9, wherein the digestion comprises addition of an agent selectedfrom the group consisting of an enzyme, a mineral acid, a carboxylicacid, a base and combinations thereof.
 12. The method according to claim9, further comprising extraction of the target by membrane filtration.13. The method of claim 6, wherein the micro-organism is selected fromthe group consisting of bacteria, bacterial spores, archea, yeast andfungi.
 14. The method of claim 6, wherein the mammalian cell is a cellfrom blood plasma.
 15. The method of claim 14, wherein the mammaliancell is selected from the group consisting of leukocytes, erythrocytesand thrombocytes.
 16. The device according to claim 1, wherein saidhandle comprises a material selected from the group consisting of wood,natural or synthetic polymeric material, plastics, and rubber materials.17. A method for sampling an area for a target comprising: i) awet-sampling step comprising swiping the surface of the area with thegelatine-based sponge of a first device according to claim 1, whereinsaid gelatine-based sponge is pre-wetted; ii) followed by a dry-samplingstep comprising swiping said surface with the gelatine-based sponge of asecond device according to claim 1, iii) transferring target collectedby said wet-sampling and dry-sampling steps to a transfer medium,thereby recovering the target from said surface.
 18. The methodaccording to claim 17, wherein said handle comprises a material selectedfrom the group consisting of wood, natural or synthetic polymericmaterial, plastics, and rubber materials.
 19. The method according toany one of claims 4 and 6 wherein said swab is pre-wetted.
 20. Themethod according to any one of claims 4, 5 and 6, wherein said targetcomprises cells and wherein said method further comprises culturingcells collected on the swab in a growth medium.